Surface stabilization of biosensors

ABSTRACT

A sensor apparatus includes a substrate, a semiconductor device disposed over the substrate, the semiconductor device having a surface electrode structure, and a saccharide coating formed over the surface electrode structure. The saccharide coating can be removed prior to use. The semiconductor device can further include a well and optionally a bead disposed in the well.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No. 15/061,273filed Mar. 4, 2016, which application claims the benefit under 35 U.S.C.§ 119(e) of U.S. Provisional Application No. 62/128,916, filed Mar. 5,2015. The entire contents of the aforementioned applications areincorporated by reference herein.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to surface stabilized biosensorsand methods of stabilizing such biosensors.

BACKGROUND

There is increasing interest in electronic sensors for detectingchemical and biological agents, analytes, or reaction byproducts.Furthermore, there is a push to develop sensors with greatersensitivity, capable of detecting ever smaller changes in concentrationor solution pH. There is also a move to decrease reaction volumes,particularly when detecting the byproducts of reactions.

In one exemplary system, pH-based sequencing of biomolecules, such asnucleic acids or proteins, utilizes small changes in pH in reactionvolumes on the order of nanoliters or smaller. For example, targetnucleic acids can be disposed in volumes of less than a nanoliter andnucleotide incorporation along the target nucleic acids can be detectedbased on small changes in pH resulting from the nucleotideincorporation.

The reliability of such sensitive sensors can be influenced by both thestability of the sensor devices and the consistency of the sensorcalibration. Such stability and consistent calibration can be affectedby packaging, storage, and transportation.

SUMMARY

In an exemplary embodiment, a sensor apparatus includes a substrate, asemiconductor device disposed over the substrate, and a flow cell lid.The semiconductor device has a surface electrode structure. A flow cellis defined between the substrate and the flow cell lid. In an example, asaccharide solution can be applied through the flow cell and over thesemiconductor device, forming a saccharide coating over the surfaceelectrode structure of the semiconductor device. The sensor apparatuscan be dried and package, leaving the saccharide coating over thesurface electrode structure during transport and storage. At a point ofuse, a wash solution can be applied through the flow cell, removing thesaccharide coating and exposing the surface electrode structure to fluidpassing through the flow cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1, FIG. 2A, FIG. 2B and FIG. 2C include illustrations of portionsof an exemplary sensor apparatus.

FIG. 3, FIG. 4, FIG. 5, and FIG. 6 include illustrations of exemplarysensor apparatuses.

FIG. 7 and FIG. 8 include illustrations of exemplary methods fortreating and using a sensor apparatus.

FIG. 9, FIG. 10, FIG. 11, FIG. 12, and FIG. 13 include illustrations ofan exemplary preparation manifold of a treatment system.

FIG. 14 includes an illustration of an exemplary treatment system.

FIG. 15 includes an illustration of an exemplary sensing system.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In an exemplary embodiment, a sensor apparatus includes a substrate, asemiconductor device disposed over the substrate, and a flow cell lid.The semiconductor device has a surface electrode structure. Thesemiconductor device can further include a well structure defining wellsover the surface electrode structures. Optionally, beads can be disposedin the wells, and the beads can be conjugated to nucleic acids. A flowcell is defined between the substrate and the flow cell lid. In anexample, a saccharide solution can be applied through the flow cell andover the semiconductor device, forming a saccharide coating over thesurface electrode structure of the semiconductor device and optionally,over a bead. The sensor apparatus can be dried and package, leaving thesaccharide coating over the surface electrode structure during transportand storage. At a point of use, a wash solution can be applied throughthe flow cell, removing the saccharide coating and exposing the surfaceelectrode structure to fluid passing through the flow cell.

In a particular embodiment, a sequencing system uses a sensor apparatusthat includes a flow cell to which a sensory array is exposed. Thesequencing system includes communication circuitry in electroniccommunication with the sensory array, and includes containers and fluidcontrols in fluidic communication with the flow cell. In an example,FIG. 1 illustrates an expanded and cross-sectional view of a flow cell100 and illustrates a portion of a flow chamber 106. A reagent flow 108flows across a surface of a microwell array 102, in which the reagentflow 108 flows over the open ends of microwells of the microwell array102. The microwell array 102 and a sensor array 105 together can form anintegrated unit forming a lower wall (or floor) of flow cell 100. Areference electrode 104 can be fluidly coupled to flow chamber 106.Further, a flow cell cover or lid 130 encapsulates flow chamber 106 tocontain reagent flow 108 within a confined region.

FIG. 2A illustrates an expanded view of a microwell 201 and a sensor214, as illustrated at 110 of FIG. 1. The volume, shape, aspect ratio(such as base width-to-well depth ratio), and other dimensionalcharacteristics of the microwells can be selected based on the nature ofthe reaction taking place, as well as the reagents, byproducts, orlabeling techniques (if any) that are employed. The sensor 214 can be achemical field-effect transistor (chemFET), more specifically anion-sensitive FET (ISFET), with a floating gate 218 having a sensorplate 220 optionally separated from the microwell interior by apassivation layer 216 or other metal, metal nitride, or metal oxidelayers, or combinations thereof. Together, the sensor plate 220,optional passivation layer 216, or other layers form an electrodestructure. The sensor 214 can be responsive to (and generate an outputsignal related to) the amount of a charge 224 present on the passivationlayer 216 opposite the sensor plate 220. Changes in the charge 224 cancause changes in a current between a source 221 and a drain 222 of thechemFET. In turn, the chemFET can be used directly to provide acurrent-based output signal or indirectly with additional circuitry toprovide a voltage-based output signal. Reactants, wash solutions, andother reagents can move in and out of the microwells by a diffusionmechanism 240.

In an embodiment, reactions carried out in the microwell 201 can beanalytical reactions to identify or determine characteristics orproperties of an analyte of interest. Such reactions can generatedirectly or indirectly byproducts that affect the amount of chargeadjacent to the sensor plate 220. If such byproducts are produced insmall amounts or rapidly decay or react with other constituents, thenmultiple copies of the same analyte can be analyzed in the microwell 201at the same time to increase the output signal generated. In anembodiment, multiple copies of an analyte can be attached to a solidphase support 212, either before or after deposition into the microwell201. The solid phase support 212 can be microparticles, nanoparticles,beads, solid or porous comprising gels, or the like. For simplicity andease of explanation, the solid phase support 212 is also referred hereinas a bead or particle. For a nucleic acid analyte, multiple, connectedcopies can be made by rolling circle amplification (RCA), exponentialRCA, or like techniques, to produce an amplicon without the use of asolid support.

Signal quality from the chemFET can be influenced by surface qualitiesof the microwell 201 and electrode structure. To protect or stabilizethe surface, a saccharide coating can be applied over the surfaces. Forexample, as illustrated in FIG. 2B, following assembly and prior to use,a saccharide coating 226 can be formed over the sensor structure 214.The saccharide coating 226 can be conformal, as illustrated in FIG. 2B.Alternatively, the saccharide coating can fill the microwell 201 andoptionally portions of the flow cell 106.

In a further example, a bead can be disposed in the well. For example,as illustrated in FIG. 2C, the saccharide coating 226 can be formed overthe bead 212 and over the sensor structure 214. The saccharide coatingcan be conformal or fill the microwell 201, and can optional occupyportions of the flow cell 106.

Prior to use, a wash solution can be applied through the flow cell 106to remove the saccharide coating 226, providing a microwell 201 intowhich, for example, a solid phase support 212 can be provided andmeasurements of ion concentration or pH can be made, or when a solidsupport is present prior to washing, the solid support can be exposedfor use in measurements.

Flow cells can be assembled with a microwell array and sensor array in avariety of ways. In one embodiment, illustrated in FIGS. 3-6, a flowcell is made by attaching a fluidic interface member to a housingcontaining a sensor chip. Typically, an integrated microwell-sensorarray (i.e., a sensor chip) is mounted in a housing or package thatprotects the chip and provides electrical contacts for communicatingwith other devices. A fluidics interface member is designed to provide acavity or flow chamber for reagents to pass through when it is sealinglyattached to such packaging. In one aspect, such attachment isaccomplished by gluing the pieces together. FIG. 3 illustrates a bottomview (or face) of a component 400 of a flow cell. In the illustratedembodiment, a complete flow cell is formed by attaching the component400 to a package containing a sensor array (as shown in FIGS. 5 and 6).A ridge 401 is elevated from a surface 413 and forms walls 410 of anellipsoidal flow chamber 408 when mated with the chip 430 illustrated inFIG. 5. The component 400 can be glued to the chip housing 430 to form afluid-tight seal. FIG. 4 illustrates a top view (or face) 416 of thecomponent or member 400 showing inlet and outlet collars 418 and 420that permit the flow cell to be sealingly connected to a fluidic system.Inlet and outlet tubes connected to elastomeric annular members can beinserted into the collars 418 and 420 so that the elastomeric materialforms a seal along the floor and walls of collars 418 and 420. Othermethods of connecting a flow cell to a fluidics system can be used,including other types of pressure fittings, clamp-based fittings,screw-on fittings, or the like. The component 400 can be adapted toaccommodate different sized chips with a simple design change, asillustrated by passages 422 and 424. Namely, for a small array 434illustrated in FIG. 5, a passage having an opening at the center of theinlet collar 418 and of the outlet collar 420 can be directed by suchpassage towards the center of the component or member 400 to an inletport and outlet port over the array 430. Likewise, for a large array436, illustrated in FIG. 6, similar passages 442 and 444 can be directedaway from the center of the component 400 and to the inlet and outlet ofthe array 436. Such a design advantageously provides a single basic flowcell design that can be used with multiple sensor array sizes. Aprotruding tab 412 and a bevel 414 can be employed to ensure correctlyoriented placement of a chip into a complementary socket or appliancefor making fluidic and electrical connections to the rest of theapparatus.

The chip or chip assembly can be treated to protect the sensors or beadsdisposed on the chip during transportation. In a particular example, asillustrated in FIG. 7, a method 700 includes applying a flow cell lidover a semiconductor substrate to define a flow cell of a sensorapparatus, as illustrated at 702. Access ports through the flow cell lidcan provide fluidic access to the flow cell sensor devices of the sensorapparatus. The sensor devices can include electrode or gate structuresthat are exposed to fluids applied through flow cell lid and flowingthrough flow cell.

As illustrated at 704, a cleaning solution can be applied through theflow cell. The cleaning solution can be a base solution, such as asolution including NaOH, an acid solution, or a combination thereof. Inan example, the acid solution is a non-aqueous solution including ananionic surfactant, such as a sulfonic acid surfactant, for example,dodecyl benzene sulfonic acid (DBSA). In particular, the semiconductorsubstrate can be cleaned using a sequence of flowing the non-aqueousacid solution, water or alcohol and the base solution.

Optionally, beads can be deposited over the sensor device, for example,in wells formed over the semiconductor substrate, as illustrated at 706.Optionally, the beads can be conjugated to a biomolecule, such as anucleic acid.

In an example, the beads can be formed from a polymer network, such as anon-nucleic acid polymer network. Exemplary non-nucleic polymer networksagarose; polyethylene glycol; polyoxybutylene; diethylacrylamide;polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; the like; or anycombination thereof. In a particular example, the polymer networkincludes polyethylene glycol. In another example, the polymer networkincludes polyacrylamide. In particular, the polymer network can form ahydrogel. For example, the bead when in an aqueous solution can have0.1% to 10% polymer by weight, such as 0.5% to 8% polymer or 0.8% to 5%polymer.

In a further example, the beads can be conjugated to a biomolecule. In aparticular example, the biomolecule can be covalently bonded to thepolymer network of the bead either directly or through a linker. Thebiomolecule can be a nucleic acid, such as DNA or RNA. In particular,the nucleic acid can be an oligonucleotide, such as a capture probe or aprimer. In another example, the nucleic acid can be a polynucleotide tobe sequenced.

In an example, as illustrated 708, a saccharide solution can be appliedthrough the flow cell. In an example, the saccharide solution is anaqueous solution that includes a saccharide. Optionally, the saccharidesolution can also include a surfactant. In a further example, thesaccharide solution can include an alcohol.

The saccharide can include a monosaccharide, a disaccharide, apolysaccharide, a derivative thereof, or a combination thereof. In anexample, a monosaccharide includes glucose, fructose, galactose, or acombination thereof. In another example, a disaccharide includessucrose, trehalose, maltose, or lactose, or a combination thereof. In aparticular example, the disaccharide includes sucrose. In anotherexample, the disaccharide includes trehalose. In a further example, thesaccharide can include hyaluronic acid. The saccharide can be includedin the saccharide solution in a range of 5 wt % to 30 wt %, such as arange of 5 wt % to 20 wt %, a range of 10 wt % to 20 wt %, or a range of10 wt % to 15 wt %.

The saccharide solution can further include a surfactant. The surfactantcan be an ionic surfactant, an amphoteric or zwitterionic surfactant, anon-ionic surfactant, or a combination thereof. The ionic surfactant canbe an anionic surfactant. An exemplary anionic surfactant includes asulfate surfactant, a sulfonate surfactant, a phosphate surfactant, acarboxylate surfactant, or any combination thereof. An exemplary sulfatesurfactant includes alkyl sulfates, such as ammonium lauryl sulfate,sodium lauryl sulfate (sodium dodecyl sulfate, (SDS)), or a combinationthereof; an alkyl ether sulfate, such as sodium laureth sulfate, sodiummyreth sulfate, or any combination thereof; or any combination thereof.An exemplary sulfonate surfactant includes an alkyl sulfonate, such assodium dodecyl sulfonate; docusates such as dioctyl sodiumsulfosuccinate; alkyl benzyl sulfonate (e.g., dodecyl benzene sulfonicacid or salts thereof); or any combination thereof. An exemplaryphosphate surfactant includes alkyl aryl ether phosphate, alkyl etherphosphate, or any combination thereof. An exemplary carboxylic acidsurfactant includes alkyl carboxylates, such as fatty acid salts orsodium stearate; sodium lauroyl sarcosinate; a bile acid salt, such assodium deoxycholate; or any combination thereof.

In another example, the ionic surfactant can be a cationic surfactant.An exemplary cationic surfactant includes primary, secondary or tertiaryamines, quaternary ammonium surfactants, or any combination thereof. Anexemplary quaternary ammonium surfactant includes alkyltrimethylammoniumsalts, such as cetyl trimethylammonium bromide (CTAB) or cetyltrimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC);polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; dioctadecyldimethylammoniumbromide (DODAB); or any combination thereof.

An exemplary amphoteric or zwitterionic surfactant includes a primary,secondary, or tertiary amine or a quaternary ammonium cation with asulfonate, carboxylate, or phosphate anion. An exemplary sulfonateamphoteric surfactant includes(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); a sultainesuch as cocamidopropyl hydroxysultaine; or any combination thereof. Anexemplary carboxylic acid amphoteric surfactant includes amino acids,imino acids, betaines such as cocamidopropyl betaine, or any combinationthereof. An exemplary phosphate amphoteric surfactant includes lecithin.In a further example, the amphoteric or zwitterionic surfactant can bean alkylamino alkylsulfonate, such as an alkyl dimethyl ammonio propanesulfonate, where the alkyl group has between 5 and 20 carbons, forexample, between 8 and 18 carbons, between 8 and 14 carbons, or between10 and 14 carbons. In a particular example, the surfactant includesn-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.

In another example, the surfactant can be a non-ionic surfactant such asa polyethylene glycol-based surfactant, an alkyl pyrrolidine surfactant,an alkyl imidazolidinone surfactant, an alkyl morpholine surfactant, analkyl imidazole surfactant, an alkyl imidazoline surfactant, or acombination thereof. In a particular example, thepolyethylene-glycol-based surfactant includes a polyethylene-glycolether, such as an alkylphenol polyethoxylate. In a further example, thenon-ionic surfactant can be an alkyl polysaccharide. In another example,the non-ionic surfactant includes a non-ionic fluorosurfactant, such asan ethoxylated fluorocarbon. In a further example, the surfactantsolution can include octyl pyrrolidine.

In particular, the surfactant solution can include combinations of suchsurfactants. For example, the surfactant solution can include acombination of a zwitterionic surfactant with an anionic surfactant. Ina particular example, the surfactant solution can include a zwitterionicsurfactant, such as an alkylamino alkylsulfonate, and an anionicsurfactant, such as a sulfate surfactant, for example SDS.

In an example, the surfactant solution can include one or moresurfactants having a total concentration in the range of 0.001% to 20%by weight. For example, surfactant can be included in a total amount ina range of 0.01% to 10.0%, such as a range of 0.01% to 5.0%, a range of0.05% to 1.0%, or a range of 0.05% to 0.5% by weight.

In a further example, the surfactant solution can include an alcohol,such as methanol, ethanol, isopropyl alcohol, isobutyl alcohol, or acombination thereof. In an example, the alcohol can be included in anamount in a range of 5.0 wt % to 70.0 wt %, such as a range of 10 wt %to 50 wt %.

As illustrated at 710, the saccharide solution can be removed from theflow cell, leaving a saccharide coating on the semiconductor substrate.The saccharide coating can be conformal. In another example, thesaccharide coating can fill the wells defined over the semiconductorsubstrate. In particular, the saccharide coating is disposed overelectrode structures of sensor devices on the semiconductor substrate.

As illustrated at 712, the sensor apparatus can be packaged. Forexample, the sensor apparatus can be dried and then inserted into asealed package for shipping. In an example, the packaging haselectrostatic dissipative characteristics. In another example, thepackaging can be water or humidity resistant. In an additional example,a desiccant can be added to the packaging. As such, the saccharidecoating remains in contact with the sensor device for transport andstorage of the sensor apparatus.

At the point of use, the saccharide coating can be removed inpreparation for utilizing the sensor apparatus. As illustrated in FIG.8, a method 800 includes flowing a wash solution through the flow cellof the sensor apparatus, as illustrated at 802. In an example, the washsolution can be water or an aqueous solution. In a further example, thewash solution can include a surfactant. In an additional example, thewash solution can include an alcohol. An exemplary alcohol includesethanol, methanol, isopropyl alcohol, isobutyl alcohol, or a combinationthereof. In an example, the alcohol can be included in an amount in arange of 5 wt % to 70 wt %, such as a range of 10% to 50%. In anotherexample, pure alcohol can be used.

The wash solution flowing through the flow cell removes the saccharidecoating and exposes a sensor electrode structure of the sensor devicesand optionally, a bead. Following removal of the saccharide coating, thesensor can be used.

For example, a biomolecular target, such as a nucleic acid or protein,can be applied to the sensor apparatus, as illustrated at 804. In aparticular example, nucleic acids or proteins can be applied over asensor electrode of the sensor device. In an example, the biomoleculartarget is attached to a bead and deposited in a well of the sensordevice. In another example, wells of the sensor device can include beadsprior to washing, and the biomolecular target can be captured by suchbeads. In a particular example, a nucleic acid target can be captured bya bead having a complementary oligonucleotide and optionally amplifiedto provide a plurality of copies of the nucleic acid target on the bead.In particular, a plurality of copies of nucleic acids can be appliedover a sensor device and sequencing-by-synthesis can be performedthrough the detection of changes in pH in response to nucleotideincorporation.

As illustrated at 806, the sensor apparatus can be applied to a detectorsystem. For example, the sensor apparatus can be connected fluidicly toa set of reagents and can be connected electronically to measurement andcontrol systems. A series of reagents can be applied to a flow cell ofthe sensor apparatus, and measurements can be made using the sensordevices of the sensor apparatus. In a particular example, as illustratedat 808, a biomolecular target can be sequenced using such a detectorsystem. For example, nucleic acids can be sequenced usingsequencing-by-synthesis and detecting changes in pH using the sensordevices.

FIG. 9, FIG. 10, FIG. 11, FIG. 12, and FIG. 13 include illustrations ofan exemplary treatment apparatus for treating sensor apparatuses inpreparation for packaging and storage. As illustrated in FIG. 9, atreatment apparatus 900 includes a deck 902 configured to secure a tray904. The tray 904 includes slots 906 to receive sensor apparatuses 908.As illustrated, the sensor apparatuses 908 are applied to haveelectronic interfaces facing upward and fluidic interfaces to the flowcell facing downward. Alternatively, the sensor apparatuses 908 can beapplied to have electronic interfaces facing downward and fluidicinterfaces to the flow cell facing downward.

Using a handle 910, the deck 902 can be guided along the rails 912 underthe clamp 914. When positioned under the clamp 914, the deck 902 alignsthe sensor devices and, in particular, the fluid access to the flowcells of the sensor apparatuses with a fluidics manifold 1022 and ports1124, as illustrated in FIG. 10 and FIG. 11.

The clamp 914 is supported by supports 918. A handle 916 can motivatethe clamp 914 downward, pushing the deck 902 down to engage the fluidmanifold 1022. An engagement structure 1020 can include one or moreresilient structures 1226 (illustrated in FIG. 12) to engage sensorapparatuses and motivate them against the fluid ports 1124.

As the handle 916 is lowered, the structure 1020 engages the platform902 and drives the deck 902 and the tray 904 of sensor apparatuses 908down. The platform 902 is driven along an axle 1228 until the fluidports of the sensor apparatuses 908 engaged the fluid ports 1124 of thefluid manifold 1022. When the handle 916 driven upward, the platform 902is driven up along axle 1228, disengaging the sensor apparatuses 908from the fluid ports 1124. The deck 902 and the sensor apparatuses 908can be pulled out from under the clamp 914 using the handle 910.

In a particular example, the manifold 900 forms part of an apparatus tofluidicly engage the fluid ports of sensor apparatuses and apply one ormore reagent solutions through flow cells of the sensor apparatuses,including a saccharide solution. For example, as illustrated in FIG. 14,a treatment system 1400 includes reagent containers 1402 connected to afluid manifold 1404. The fluid manifold 1404 is fluidicly connected tosubmanifolds 1406 that can each, through the use of the treatmentapparatus 900 apply fluid through flow cells of sensor apparatuses 1408.In a particular example, fluid can be driven or drawn from the reagentcontainers 1402 by pressurizing the containers or through the use ofpumps drawing fluid from the containers 1402. The manifold 1404 canselectively flow reagents from the reagent containers 1402 to thesubmanifolds 1406. The fluid can then flow through the manifold 1404 andthe submanifold 1406 through the flow cells of sensor apparatuses 1408based on the control of the downstream valves 1410. When one of thevalves 1410 is open, the fluid can flow through the flow cells of sensorapparatuses 1408 and into the waste container 1412.

In an example, each of the valves 1410 can be open simultaneouslyallowing continuous flow through the system. Alternatively, sensorapparatuses can be treated individually by selectively flowing a reagentthrough the manifold 1404 and submanifolds 1406 and selectively throughone or more of the sensor apparatuses 1408 based on the position of thevalves 1410 downstream of the sensor apparatuses 1408.

For example, the system can include wash solution, acid or base cleaningtreatment solutions, and a saccharide solution as described above. Forexample, a wash solution can be applied over one or more sensorapparatuses 1408. The sensor apparatuses can include beads in wells orcan be free of beads. A sensor apparatus can be treated using an acid orbase treatment solution. In an example, the pretreatment of the sensorapparatuses can influence concentrations of surface hydroxyl groups,influencing signal associated with pH measurement. The surface can bestabilized by applying the saccharide solution through the flow cell thesensor apparatuses and leaving a saccharide coating over the sensorelectrodes of the sensor devices and optionally, over a bead. The sensorapparatuses 1408 can be removed from the treatment apparatus, dried, andpackaged, ready for transportation and storage.

As described above, surface stabilized biosensors can be washed and usedto detect biomolecular targets or associated byproducts. In particular,the biosensors, e.g., the sensor apparatuses, can be washed as describedabove and applied to a detection system, such as a sequencing system.

In an example, beads or particles to which biomolecules can be or areattached can be applied to the biosensors for determiningcharacteristics of the biomolecules. The beads can be applied followingwashing the saccharide coating or prior to treatment with the saccharidesolution. In particular, the biosensors can be used for sequencingnucleic acid or protein target sequences conjugated to the amplifiedbeads or particles. For example, sequencing can include label-free DNAsequencing, and in particular, pH-based DNA sequencing. Beads orparticles including DNA templates and having a primer and polymeraseoperably bound are loaded into reaction chambers (such as microwells ofthe sensor apparatus), after which repeated cycles of deoxynucleosidetriphosphate (dNTP) addition and washing are carried out. Alternatively,a bead can be present prior to washing, and target nucleic acids can beapplied to the bead. Such templates are typically attached as clonalpopulations to the bead or particle, and such clonal populations areloaded into reaction chambers. In each addition step of the cycle, thepolymerase extends the primer by incorporating added dNTP when the nextbase in the template is the complement of the added dNTP. When there isone complementary base, there is one incorporation, when two, there aretwo incorporations, when three, there are three incorporations, and soon. With each such incorporation a hydrogen ion is released, andcollectively a population of templates releases hydrogen ions causingvery slight changes the local pH of the reaction chamber which isdetected by an electronic sensor.

FIG. 15 diagrammatically illustrates an apparatus for carrying outpH-based nucleic acid sequencing. Each electronic sensor of the sensorapparatus generates an output signal that depends on the value of areference voltage. In FIG. 15, a housing 600 containing a fluidicscircuit 602 is connected by inlets to reagent reservoirs 614, to a wastereservoir 620 and to a flow cell 634 by a passage 632 that connects afluidics node 630 to an inlet 638 of the flow cell 634. Reagents fromthe reservoirs 614 can be driven to the fluidic circuit 602 by a varietyof methods including pressure, pumps, such as syringe pumps, gravityfeed, and the like, and are selected by control of valves 650. A controlsystem 618 includes controllers for valves 650 that generate signals foropening and closing via electrical connection 616. The control system618 also includes controllers for other components of the system, suchas wash solution valve 624 connected thereto by control line 622, and areference electrode 628. The control system 618 can also include controland data acquisition functions for the flow cell 634. In one mode ofoperation, the fluidic circuit 602 delivers a sequence of selectedreagents (1, 2, 3, 4, or 5) to the flow cell 634 under programmedcontrol of the control system 618, such that in between selected reagentflows, the fluidics circuit 602 is primed and washed, and the flow cell634 is washed. Fluids entering the flow cell 634 exit through an outlet640 and are deposited in a waste container 636. Throughout such anoperation, the reactions or measurements taking place in the flow cell634 have a stable reference voltage because the reference electrode 628has a continuous, i.e. uninterrupted, electrolyte pathway with the flowcell 634, but is in physical contact with only the wash solution.

EXAMPLES Example 1

pH-based sequencing apparatuses (Ion Torrent™ Proton I) are treated withdifferent solutions including a saccharide or polymer.

The apparatuses are prepared by flowing a series of wash solutionsthrough the flow cells of the apparatuses. A solution including 5 wt %dodecyl benzene sulfonic acid (DBSA) in undecane is flowed through theflow cell for 1 minute. Isopropyl alcohol is flowed through the flowcell to remove the DBSA in undecane. Water is applied through the flowcell followed by a 10 mM NaOH solution for 2:45 min. Water is appliedthrough the flow cell followed by isopropyl alcohol (IPA) andsubsequently nitrogen gas.

Following drying under nitrogen, an aqueous solution including 10 wt %or 20 wt % of a selected saccharide or polymer is applied through theflow cell. Controls are not treated. The saccharides are selected fromsucrose, galactose, and trehalose. The polymer is selected from apolyethylene glycol polymer (PEG 200 or PEG 1000), polypropylene glycol,an ethylene oxide/propylene oxide block copolymer (Pluronic P65), and apolyol (Pluronic F127). Following application of the solution, the flowcell is flushed with nitrogen. The apparatuses' surfaces are observedfor uniformity of coating. The apparatuses are further test for initialbead loading uniformity and for bead loading uniformity after aging(heating at 70° C. for 7 days). Bead loading is performed on an IonTorrent™ Ion Chef™ using Ion Torrent™ kits incorporating the above chipapparatus.

Those apparatuses treated with sucrose, galactose, and trehalosedemonstrate desirable uniformity and both initial and aged bead loading,providing improvement over the control. The polyol polymer demonstratessimilar improvement relative to the controls. The ethyleneoxide/propylene oxide copolymer solution provides similar to slightlybetter bead loading relative to the control. The PEG and propyleneglycol polymers provide less desirable bead loading than the control.

Example 2

pH-based sequencing apparatuses (ION Torrent Proton I) are treated withsaccharide solutions incorporating different surfactants.

The apparatuses are prepared by flowing a series of wash solutionsthrough the flow cells of the apparatuses. A solution including 5 wt %dodecyl benzene sulfonic acid (DBSA) in undecane is flowed through theflow cell for 1 minute. Isopropyl alcohol is flowed through the flowcell to remove the DBSA in undecane. Water is applied through the flowcell followed by a 10 mM NaOH solution for 2:45 min. Water is appliedthrough the flow cell followed by isopropyl alcohol (IPA) andsubsequently nitrogen gas.

Following drying under nitrogen, an aqueous saccharide solutionincluding 10 wt % or 20 wt % sucrose and between 0.05 wt % and 0.2 wt %of a select surfactant is applied through the flow cell. The surfactantis selected from 0.05 wt % of a non-ionic fluorosurfactant (Thetawet8150), 0.02 wt % of a non-ionic surfactant (Triton X-100), 0.1 wt % ofan alkyl polysaccharide surfactant (Multitrope), 0.2 wt % dioctyl sodiumsulfosuccinate, or 0.2% of a zwitterionic surfactant (Anzergent 3-12).Following application of the saccharide solution, the flow cell isflushed with nitrogen. The apparatuses' surfaces are observed foruniformity of coating.

The saccharide solutions including 10 wt % sucrose provided a moreuniform coating and less streaking than the saccharide solutionsincluding 20 wt % sucrose. Each of the surfactants had similar effectson uniformity. But, the Triton X-100 surfactant produced beads ofsucrose on a lid of the flow cell.

Example 3

To assess the stability of pre-loaded cassettes on Ion Torrent™ 541chips, the chips (apparatuses) are pre-loaded with polyacrylamide beads,coated using a sucrose solution, and vacuum sealed with desiccant in afoil pouch.

A set of 16 kits including Ion Torrent 541 chips (apparatuses) andassociated polyacrylamide beads are loaded by spinning a solutionbearing the beads on the apparatuses for 2 minutes, foam scraping, andflushing and vacuuming. The beads are conjugated to oligonucleotidesthat act to capture target polynucleotides and extend to makecomplementary copies of the target polynucleotides.

Half of the apparatuses are coated with a sucrose solution including1431 g H₂O, 358 g sucrose, and 3.5 g Anzergent 3-12. Each of theapparatuses is placed in a tray and vacuum sealed in a foil pouch forstorage.

Apparatuses are tested at 1 day, 3 days, and 10 days of storage. Theapparatuses are washed to remove the sucrose coating and subjected toamplification to yield beads conjugated to polynucleotides, which aresequenced.

Day 1 apparatuses whether sucrose coated or not demonstrate similarsequencing performance characterized by similar loading percentage, keysignal, and q20 mean performance. But, Day 3 apparatuses not stored witha sucrose coating demonstrate a significantly reduced sequencingperformance, whereas Day 3 apparatuses stored with a sucrose coatingmaintain sequencing performance. Day 10 apparatuses stored with asucrose coating also maintain sequencing performance.

In a first aspect, a sensor apparatus includes a substrate, asemiconductor device disposed over the substrate, the semiconductordevice having a surface electrode structure, and a saccharide coatingformed over the surface electrode structure.

In an example of the first aspect, the sensor apparatus further includesa well structure defined over the semiconductor device and defining awell exposing the surface electrode structure, the saccharide coatingformed at least partially within the well.

In an additional example of the first aspect and the above examples, theapparatus further includes a bead disposed in the well, the saccharidecoating formed over the bead. In an example, the bead is conjugated to anucleic acid. In another example, the bead is a hydrogel bead. In afurther example, the bead comprises a polymer selected from the groupconsisting of agarose; polyethylene glycol; polyoxybutylene;diethylacrylamide; polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; and any combinationthereof. In a particular example, the polymer is polyacrylamide.

In another example of the first aspect and the above examples, thesensor apparatus further includes a flow cell lid disposed over thesubstrate and defining a flow cell between the substrate and the flowcell lid. The surface electrode structure is accessible through the flowcell.

In a further example of the first aspect and the above examples, thesemiconductor device includes a field effect transistor having a gateelectrode structure. The surface electrode structure forms at least partof the gate electrode structure.

In an additional example of the first aspect and the above examples, thesurface electrode structure includes a conductive structure and a sensorlayer disposed over the conductive structure. For example, the sensorlayer includes a ceramic layer.

In another example of the first aspect and the above examples, thesurface electrode structure includes a floating gate structure.

In a further example of the first aspect and the above examples, thesaccharide coating includes a monosaccharide, a disaccharide, apolysaccharide, a derivation thereof, or a combination thereof. Forexample, the monosaccharide can include glucose, fructose, galactose, ora combination thereof. In an additional example, the disaccharideincludes sucrose, trehalose, maltose, or lactose. For example, thedisaccharide includes sucrose. In another example, the disaccharideincludes trehalose. In a further example, the saccharide includeshyaluronan.

In a second aspect, a sensor apparatus includes a semiconductorsubstrate, semiconductor devices formed on the semiconductor substrate,a well structure formed over the semiconductor devices and defining awell, wherein the semiconductor devices include a field effecttransistor having a gate structure responsive to a condition within thewell, and a saccharide coating disposed within the well and over thegate structure.

In an example of the second aspect, the sensor apparatus furtherincludes a flow cell lid disposed over the semiconductor substrate andthe well structure and defining a flow cell between the substrate andthe flow cell lid, the well exposed to the flow cell.

In another example of the second aspect and the above examples, theapparatus further includes a bead is disposed in the well, thesaccharide coating disposed over the bead. In an example, the bead isconjugated to a nucleic acid. In another example, the bead is a hydrogelbead. In an further example, the bead comprises a polymer selected fromthe group consisting of agarose; polyethylene glycol; polyoxybutylene;diethylacrylamide; polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; and any combinationthereof. In a particular example, the polymer is polyacrylamide.

In another example of the second aspect and the above examples, the gatestructure includes a conductive structure and a sensor layer disposedover the conductive structure. For example, the sensor layer includes aceramic layer.

In a further example of the second aspect and the above examples, thegate structure includes a floating gate structure.

In an additional example of the second aspect and the above examples,the saccharide coating includes a monosaccharide, a disaccharide, apolysaccharide, a derivation thereof, or a combination thereof. Forexample, the monosaccharide includes glucose, fructose, galactose, or acombination thereof. In another example, the disaccharide includessucrose, trehalose, maltose, or lactose. For example, the disaccharideincludes sucrose. In a further example, the disaccharide includestrehalose. In an additional example, the saccharide includes hyaluronan.

In a third aspect, a method of sequencing includes flowing a washsolution over a sensor apparatus. The sensor apparatus includes asubstrate, a semiconductor device disposed over the substrate, thesemiconductor device having a surface electrode structure, and asaccharide coating formed over the surface electrode structure. The washsolution removes the saccharide coating. The method further includesapplying nucleic acid targets to the sensor apparatus, applying thesensor apparatus to a sequencing system, and sequencing the nucleic acidtargets using the sequencing system.

In an example of the third aspect, the saccharide coating includes amonosaccharide, a disaccharide, a polysaccharide, a derivation thereof,or a combination thereof. For example, the monosaccharide includesglucose, fructose, galactose, or a combination thereof. In anotherexample, the disaccharide includes sucrose, trehalose, maltose, orlactose. In an example, the disaccharide includes sucrose. In anotherexample, the disaccharide includes trehalose. In a further example, thesaccharide includes hyaluronan.

In an additional example of the third aspect and the above examples, theapparatus further includes a well structure defining a well over thesurface electrode structure, and the apparatus further includes a beaddisposed in the well. Applying nucleic acid targets to the sensorapparatus includes contacting the nucleic acid targets with the bead. Inan example, the bead includes an oligonucleotide complementary to aportion of the nucleic acid target, and the method further includesamplifying the nucleic acid target to form copies of the nucleic acidtarget on the bead. For example, the bead is a hydrogel bead. In anexample, the bead comprises a polymer selected from the group consistingof agarose; polyethylene glycol; polyoxybutylene; diethylacrylamide;polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; and any combinationthereof. In a particular example, the polymer is polyacrylamide.

In a fourth aspect, a method of preparing a sensor apparatus includesapplying a cleaning solution through a flow cell lid over asemiconductor substrate defining a flow cell, the semiconductorsubstrate including a plurality of field effect transistors eachincluding a gate structure exposed to the flow cell, applying asaccharide solution through the flow cell, the solution including asaccharide, and removing the saccharide solution from the flow cell, alayer of the saccharide remaining over the gate structures.

In an example of the fourth aspect, applying the wash solution includesflowing a basic solution through the flow cell prior to applying thesolution including the saccharide.

In an additional example of the fourth aspect and the above examples, awell structure defines a well over the gate structure, and the methodfurther includes applying a bead into the well prior to applying thesaccharide solution. In an example, the bead is conjugated to a nucleicacid. In another example, the bead is a hydrogel bead. In a furtherexample, the bead comprises a polymer selected from the group consistingof agarose; polyethylene glycol; polyoxybutylene; diethylacrylamide;polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; and any combinationthereof. For example, the polymer is polyacrylamide.

In another example of the fourth aspect and the above examples, applyingthe wash solution includes flowing a non-aqueous surfactant solutionthrough the flow cell prior to applying the solution including thesaccharide. For example, the non-aqueous surfactant solution includes asulfonic acid surfactant in a non-aqueous solvent.

In a further example of the fourth aspect and the above examples, thesaccharide includes a monosaccharide, a disaccharide, a polysaccharide,a derivation thereof, or a combination thereof. For example, themonosaccharide includes glucose, fructose, galactose, or a combinationthereof. In an additional example, the disaccharide includes sucrose,trehalose, maltose, or lactose. For example, the disaccharide includessucrose. In another example, the disaccharide includes trehalose. Inanother example, the saccharide includes hyaluronic acid.

In an additional example of the fourth aspect and the above examples,the saccharide solution includes the saccharide in a range of 5 wt % to30 wt %.

In another example of the fourth aspect and the above examples, thesaccharide solution includes a surfactant in a range of 0.01 wt % to10.0 wt %. For example, the surfactant can include a zwitterionicsurfactant. In another example, the surfactant includes a non-ionicsurfactant.

In a fifth aspect, a method of preparing a sensor apparatus includesapplying a flow cell lid over a semiconductor substrate to define a flowcell, the semiconductor substrate including a plurality of field effecttransistors each including a gate structure exposed to the flow cell,applying a solution through the flow cell, the solution including asaccharide, and removing the solution from the flow cell, a layer of thesaccharide remaining over the gate structures.

In an example of the fifth aspect, the method further includes flowing abasic solution through the flow cell prior to applying the solutionincluding the saccharide.

In another example of the fifth aspect and the above examples, a wellstructure defines a well over the gate structure, and the method furtherincludes applying a bead into the well prior to applying the saccharidesolution. For example, the bead is conjugated to a nucleic acid. In anexample, the bead is a hydrogel bead. In another example, the beadincludes a polymer selected from the group consisting of agarose;polyethylene glycol; polyoxybutylene; diethylacrylamide;polyoxyethylene; polyacrylamide; polyoxypropylene;N,N-polydimethylacrylamide; poly(N-isopropylacrylamide);polyvinylpyrrolidone; poly-N-hydroxyacrylamide; and any combinationthereof. In a particular example, the polymer is polyacrylamide.

In another example of the fifth aspect and the above examples, themethod further includes flowing a non-aqueous surfactant solutionthrough the flow cell prior to applying the solution including thesaccharide. For example, the non-aqueous surfactant solution includes asulfonic acid surfactant in a non-aqueous solvent.

In a further example of the fifth aspect and the above examples, thesaccharide coating includes a monosaccharide, a disaccharide, apolysaccharide, a derivation thereof, or a combination thereof. Forexample, the monosaccharide includes glucose, fructose, galactose, or acombination thereof. In an example, the disaccharide includes sucrose,trehalose, maltose, or lactose. For example, the disaccharide includessucrose. In another example, the disaccharide includes trehalose. In anadditional example, the saccharide includes hyaluronic acid.

In an additional example of the fifth aspect and the above examples, thesaccharide solution includes the saccharide in a range of 5 wt % to 30wt %.

In another example of the fifth aspect and the above examples, thesaccharide solution includes a surfactant in a range of 0.01 wt % to10.0 wt %. For example, the surfactant includes a zwitterionicsurfactant. In another example, the surfactant includes a non-ionicsurfactant.

In an sixth aspect, a treatment system includes a plurality of reagentvessels, a manifold in fluidic communication with the plurality ofreagent vessels, and a treatment apparatus including a slidable deck toreceive sensor apparatuses, a clamp having resilient apparatus guides,and an access manifold in fluidic communication with the manifold andincluding ports to fluidicly connect with flow cells of the sensorapparatuses, when the slidable deck is positioned under the clamp, theclamp movable to motivate the slidable deck downward to engage theaccess manifold.

In an example of the sixth aspect, the slidable deck is to receive thesensor apparatuses with flow cell ports of the sensor apparatuses facingdown, the resilient apparatus guides to engage a side of the sensorapparatuses opposite the flow cell ports.

Such methods and devices formed using such methods provide technicaladvantages relating to improved measurement quality. It has beendiscovered that signal quality can variably degrade, depending uponfactors associated with transportation and storage. Reducing thevariability in signal quality provides for a more uniform signalmeasurement that can lead to more accurate sequence detection.

In particular, pH-based sensor devices, such as those used in pH-basedsequencing, are sensitive to factors associated with transportation andstorage. It is believed that surface groups, such as surface hydroxylgroups, influence signal quality and that the concentration of suchsurface groups can be influenced by the factors associated withpackaging, transportation, and storage, such as temperature andhumidity. The proposed system and methods provides for less variabilityis signal quality.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A method of sequencing, the method comprising:flowing a wash solution over a sensor apparatus, the sensor apparatuscomprising: a substrate; a semiconductor device disposed over thesubstrate, the semiconductor device having a surface electrodestructure; a well structure defining a well over the surface electrodestructure, a bead disposed in the well; and a saccharide coating formedover the surface electrode structure and the bead; the wash solutionremoving the saccharide coating; applying nucleic acid targets to thesensor apparatus; applying the sensor apparatus to a sequencing system;and sequencing the nucleic acid targets using the sequencing system. 2.The method of claim 1, wherein the saccharide coating includes amonosaccharide, a disaccharide, a polysaccharide, a derivation thereof,or a combination thereof.
 3. The method of claim 2, wherein themonosaccharide includes glucose, fructose, galactose, or a combinationthereof.
 4. The method of claim 2, wherein the disaccharide includessucrose, trehalose, maltose, or lactose.
 5. The method of claim 4,wherein the disaccharide includes sucrose.
 6. The method of claim 2,wherein the saccharide includes hyaluronan.
 7. The method of claim 1,wherein applying nucleic acid targets to the sensor apparatus includescontacting the nucleic acid targets with the bead.
 8. The method ofclaim 7, wherein the bead includes an oligonucleotide complementary to aportion of the nucleic acid target, the method further comprisingamplifying the nucleic acid target to form copies of the nucleic acidtarget on the bead.
 9. The method of claim 7, wherein the bead is ahydrogel bead.
 10. The method of claim 7, wherein the bead comprises apolymer selected from the group consisting of agarose; polyethyleneglycol; polyoxybutylene; diethylacrylamide; polyoxyethylene;polyacrylamide; polyoxypropylene; N,N-polydimethylacrylamide;poly(N-isopropylacrylamide); polyvinylpyrrolidone;poly-N-hydroxyacrylamide; and any combination thereof.